The major cell surface glycoprotein fibronectin is decreased after malignant transformation and is involved in cellular adhesion. We have investigated its mechanism of action. Polypeptide structural domains containing specific binding sites for the plasma membrane, collagen, heparin, and actin have been isolated, purified, and characterized biochemically and in assays for cell adhesion. The immunofluorescence localization of fibronectin was compared with known adhesive structures, and it was found to be present in close, but not in most focal, cell-substrate adhesive contact sites. A positive regulatory role in embryonic differentiation was suggested in experiments with neural crest cells, in which treatment with exogenous fibronectin promoted adrenergic cell differentiation. Our objectives will be to determine the composition and structure of the multiple active sites of fibronectin, the mechanisms by which this major cell surface protein helps to regulate cell behavior and differentiation, and the structure and function of its gene.